Gene therapy for cardiomyopathy

ABSTRACT

This invention enables the repair of cardiac function by noninvasive administration of an HGF gene in the form of Sendai virus (HVJ)-liposome into the affected cardiac muscle, thereby inducing angiogenesis of the cardiac muscle layer and repressing fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the National Stage of International Application No.PCT/JP00/06947, filed Oct. 5, 2000, which claims priority from JapanPatent Application Number 11/288532, filed Oct. 8, 1999.

TECHNICAL FIELD

The present invention relates to a method of gene therapy for treatingmyocardiopathy by noninvasive administration of an HGF (hepatocytegrowth factor) gene and therapeutic agents used therefor. Morespecifically, the present invention relates to a method of gene therapyfor treating myocardiopathy by noninvasive administration of an HGF geneinto the cardiac muscle, especially to a method of gene therapy thatmore efficiently treats heart disease, such as cardiomyopathy, anginapectoris and heart failure, by injecting an HGF gene into the affectedpart of cardiac muscle under the usage of echo, and to therapeuticagents used therefor. Moreover, the present invention relates to amethod of gene therapy which is applicable to genes other than HGF genesand that consists of administering genes to the affected part of tissuenoninvasively under the usage of echo.

BACKGROUND ART

In spite of the recent striking technical improvements in the medicalfield, many problems remain unsolved. The problem of myocardiopathy isone of the important unsolved subjects.

Myocardiopathy is a general name for diseases attributable to organicand functional abnormalities of the cardiac muscle. For example,cardiomyopathy is classified into secondary cardiomyopathy, which occursin sequence to hypertension, dysbolism, ischemic disease and such, andidiopathic cardiomyopathy (ICM), which occurs without any distinctfundamental disease. Hypertrophic cardiomyopathy (HCM) is classified asan ICM, whose cause of disease is most revealed at the genetic level. Inhalf the numbers of patients which HCM, familial history followingautosomal dominant heredity is recognized. Linkage analysis of suchfamily lines, with multiple patients as the object, revealed 5 causalloci so far and the causal gene itself is specified in 4 of them.

Many cases of dilated cardiomyopathy (DCM) occur independently, butfamilial history is recognized in 20% of the cases. Linkage analysis ofsuch family lines, with multiple patients as the object, revealed 7types of causal loci (causal genes are unknown).

Regarding myocardiopathy, research is in progress to specify causal geneand to reveal the mechanism underlying the start of disease. So far, noconcrete action for gene therapy has been done.

On the other hand, the rapid progress lately in molecular biology hasmade is possible to activate cellular function by gene transfer methodsand various attempts have been made. In particular, there are somereports for gene transfer methods to the heart, like intravenous drip(J.Clin.Invest., 90, 626–630 (1992)), direct injection (Circulation, 82,2217–2221 (1990); Circulation, 90, 2414–2424 (1994)) or coronarydiffusional infusion method that utilizes the plasmid as it is(J.Thorac.Carduivasc.Surg., 109, 716–720 (1995)) and so on, but were farfrom noninvasive concrete treatment.

DISCLOSURE OF THE INVENTION

The object of this invention is to provide a noninvasive treatment formyocardiopathy, for which effective treatment is currently unknown, andtherapeutic agents used therefor. That is, the present invention relatesto a method of gene therapy for treating myocardiopathy by noninvasiveadministration of an HGF gene and therapeutic agents used therefor. Morespecifically, the present invention relates to a method of gene therapyfor treating myocardiopathy by noninvasive administration of an HGF geneinto the cardiac muscle, especially to a method of gene therapy fortreating myocardiopathy that more efficiently treat a heart disease,such as cardiomyopathy, angina pectoris and heart failure, by injectingan HGF gene to the affected part of cardiac muscle under the usage ofecho, and to therapeutic agents used therefor. Moreover, the presentinvention relates to a method of gene therapy which is applicable togenes other than HGF genes and that consists of administering genes tothe part of affected tissue noninvasively under the usage of echo.

Present inventors investigated to find out that effective results areobtained by using an HGF gene as the gene and noninvasively infusingdirectly to the affected part of cardiac muscle layer. That is, presentinventors found out that it is effective to infuse HGF gene to theaffected part of cardiac muscle optically using echo without incision ofthe affected part or thoracotomy. Since this method is a noninvasivetreatment, it is possible to administer the present gene repeatedly,according to the condition, and therefore it is possible to treatmyocardiopathy efficiently.

Present inventors newly discovered that effective treatments can be doneby infusing genes to the affected part optically using echo and showedthat the method of the present invention enables genetic treatment ofvarious organ-specific disease.

For example, in the case where the HGF gene is used, according to thepresent invention, it is possible to treat various organ-specificdiseases like pulmonary fibrosis, cirrhosis, hepatic fibrosis and so on.Furthermore, genes other than the HGF gene are also effective in themethod of the present invention above.

Thus, the outline of the present invention is as follows:

(1) a therapeutic agent for myocardiopathy used for noninvasiveadministration comprising a hepatocyte growth factor (HGF) gene as theeffective ingredient;

(2) the therapeutic agent of (1), which is used for administration ofthe HGF gene into the cardiac muscle;

(3) the therapeutic agent of (1) or (2), wherein the HGF gene is in theform of Sendai virus (HVJ)-liposome;

(4) the therapeutic agent of (2) or (3), which is used for noninvasiveadministration to the affected part of the cardiac muscle under theusage of echo;

(5) the therapeutic agent of any of (1) to (4), which is to beadministered at least 8 times, once a week;

(6 ) the therapeutic agent of any of (1) to (5), wherein at least 10 μgof the HGF gene is used;

(7) the therapeutic agent of any of (1) to (6), wherein themyocardiopathy is selected from the group consisting of cardiomyopathy,angina pectoris and heart failure;

(8) a gene therapy agent used for noninvasive administration of a geneinto an affected part of a tissue under the usage of echo, whichcomprises genes effective for the treatment of a disorder as theeffective ingredient;

(9) the gene therapy agent of (8), wherein the affected part of thetissue is the cardiac muscle;

(10) the gene therapy agent of (8) or (9), wherein the gene is an HGFgene;

(11) a method for gene therapy for myocardiopathy, which comprises thenoninvasive administration of an HGF gene into the cardiac muscle of amammal, including a human;

(12) the method for gene therapy of (11), wherein the HGF gene is in theform of Sendai virus (HVJ)-liposome;

(13) the method for gene therapy of (11) or (12), wherein the HGF geneis administered noninvasively to a part of an affected cardiac muscleunder the usage of echo;

(14) the method for gene therapy of any of (11) to (13), wherein the HGFgene is administered at least 8 times, once per week;

(15) the method for gene therapy of any of (11) to (14), wherein themyocardiopathy is selected from the group consisting of cardiomyopathy,angina pectoris and heart failure;

(16) a method for gene therapy, which comprises the noninvasiveadministration of genes effective for the treatment of a disorder intoan affected part of a tissue under the usage of echo;

(17) the method of gene therapy of (16), wherein the affected tissue isthe cardiac muscle;

(18) the method for gene therapy of (16) or (17), wherein the gene is anHGF gene;

(19) use of an HGF gene for the production of a therapeutic agent formyocardiopathy used for noninvasive administration;

(20) the use of (19), wherein the HGF gene is in the form of Sendaivirus (HVJ)-liposome;

(21) the use of (19) or (20), wherein the therapeutic agent is atherapeutic agent used for the noninvasive administration of the HGFgene to an affected part of the cardiac muscle under the usage of echo;

(22) the use of any of (19) to (21), wherein the myocardiopathy isselected from the group consisting of cardiomyopathy, angina pectorisand heart failure;

(23) use of a gene for the production of a gene therapy agent used forthe noninvasive administration of genes effective for the treatment of adisorder into an affected part of a tissue under the usage of echo;

(24) the use of (23), wherein the affected tissue is cardiac muscle; and

(25) the use of (23) or (24), wherein the gene is an HGF gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that gene transfer under usage of echo ispossible. It is proven by the high activity rate of luciferase incardimyopathy guinea pig, in which luciferase as the reporter gene isintroduced to the heart using HVJ.

FIG. 2 is a graph showing the result of a comparison between an HGF geneand a control by measuring cardiac capillary vessel density by ALP(alkaline phosphatase) staining.

FIG. 3 is a graph showing the result of a comparison of the amount ofcardiac bloodstream between an HGF gene group, a control group and anon-treated group by evaluation with a laser Doppler imager (LDI).

FIG. 4 is a graph showing the result of a comparison of the distributiondensity of fibrosis of the heart by measurement using Masson staining.

BEST MODE FOR CARRYING OUT THE INVENTION

As used herein, “HGF gene” means a gene that can express HGF (the HGFprotein). Such genes include genes with deletion of a part of the genesequence, substitution by another base of the gene sequence, insertionof other base sequence, or binding of bases to the 5′ terminus and/or 3′terminus, so long as the expressed polypeptide thereof has substantiallythe same effect as HGF. For example, HGF genes described in Nature342:440 (1989); Japanese Patent No., 2777678;Biochem.Biophys.Res.Commun. 163:967(1989); andBiochem.Biophys.Res.Commun. 172:321(1990) are included. These genes canbe used in the present invention.

The base sequence of the HGF gene (the cDNA encoding HGF) of the presentinvention has been described in the above literature and is alsoregistered with databases, such as Genbank. Thus, based on such sequenceinformation, a suitable DNA portion is used as a PCR primer; forexample, by performing an RT-PCR reaction on mRNA derived from the liveror leukocytes, cDNA of HGF can be cloned. Such cloning can easily beperformed by a person skilled in the art according to a basic textbook,such as Molecular Cloning 2nd Ed. Cold Spring Harbor Laboratory Press(1989). Modification and such of the HGF gene can be also readily doneby a person skilled in the art according to the above basic textbook.

Subsequently, methods of gene transfer, dosage forms, dose and the likefor use in gene therapy of the present invention are explained.

The dosage form of a gene therapy agent comprising the above gene as aneffective ingredient to be administered to patients are roughlyclassified into two groups: one is the case in which a nonviral vectoris used, and the other is in which a viral vector is used. Methods forpreparation and administration thereof are explained in detail inexperimental manuals (Supplement of Experimental Medicine, BasicTechnology in gene therapy, Yodosha (1996); Supplement of ExperimentalMedicine, Experimental Methods in Gene Introduction and ExpressionAnalysis, Yodosha (1997); Handbook for Development and Research of GeneTherapy, Japan Society of Gene Therapy ed., NTS (1999)). Specifics areexplained below.

A. Usage of a Nonviral Vector

A recombinant expression vector, in which a gene of interest has beenintegrated into a commonly used gene expression vector, may be used tointroduce the gene of interest into cells or tissue by the followingmethod etc.

Illustrative methods of gene transfer into cellsinclude the liopfectionmethod, calcium phosphate co-precipitation method, DEAE-dextran method,direct DNA introduction methods using micro glass tubes, and the like.

Regarding methods of gene transfer into the tissue, the recombinantexpression vector may be incorporated into the cell by subjecting it toany method, such as the gene transfer method with internal typeliposome, method of gene introduction with electrostatic type liposome.HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposomemethod), receptor-mediated gene introduction method, method ofintroducing DNA molecules together with carriers (metal particles) by aparticle gun, method of directly introducing naked-DNA, method ofintroduction with positively-charged polymers and the like.

Among them, the HVJ-liposome is a fusion product prepared by enclosing aDNA into a liposome made of lipid bilayer, which is fused to inactivatedSendai virus (Hemagglutinating virus of Japan: HVJ). The HVJ-liposomemethod is characterized by very high fusing activity with the cellmembrane as compared to the conventional liposome method, and is apreferred mode of introduction. For the method of preparingHVJ-liposome, see, the literature for details. (Separate volume ofExperimental Medicine, Basic Technology in gene therapy, Yodosha (1996);experimental Methods in Gene Introduction and Expression Analysis,Yodosha (1997); J.Clin.Invest. 93:1458–1464 (1994); Am.J.Physiol.271:R1212–1220 (1996)) and the like, and experimental examples describedbelow for details.

In particular, the Z strain (available from ATCC) is preferred as theHVJ strain, but other HVJ strains (for example, ATCC VR-907 and ATCCVR-105) may also be used.

Furthermore, the method of directly introducing naked-DNA is the mostsimple method among the methods describer above, and in this regard apreferred method of introduction.

Expression vectors as used herein may be any expression vectors so longas they permit the in vivo expression of the gene of interest. Examplesinclude expression vectors such as pCAGGS (Gene 108:193–200 (1991)),pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stratagene) and the like.

B. Usage of a Viral Vector

Representative methods that use viral vectors include those using viralvectors such as recombinant adenovirus, retrovirus and the like. Morespecifically, the gene of interest can be introduced into a DNA or RNAvirus such as detoxified retrovirus, adenovirus, adeno-associated virus,herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus,Sendai virus, SV40, human immunodeficiency virus (HIV) and the like,which is then infected to the cell to introduce the gene into the cell.

Among the above viral vectors, the efficiency of infection of adenovirusis known to be much higher than that of other viral vectors. In thisregard, it is preferred to use an adenovirus vector system.

As methods of introducing a gene therapy agent into a patient, there arein vivo methods, which permit direct introduction of the gene therapyagent into the body, and ex vivo methods, in which certain cells areremoved from human, to which the gene therapy agent is introduced andwhich are returned into the body thereafter (Nikkei Science, April 1994issue pp. 20–24; Monthly Yakuji, 36(1): 23–48 (1994); Supplement ToExperimental Medicine 12 (15) (1994); Handbook for Development andResearch of Gene Therapy, NTS (1999)). According to the presentinvention, the in vivo method is preferred.

Dosage forms may take various forms according to various administrationregimens described above (for example, liquids). When, for example, aninjection containing the gene as an effective ingredient is to be used,said injection may be prepared by dissolving the effective ingredient(s)into a standard solvent (a buffer such as PBS, physiological saline,sterile water, etc.). The injection liquid may then be filter-sterilizedwith filter as needed and then filled into sterilized containers.Conventional carriers and so on may be added to the injection.Liposomes, such as HVJ-liposome, may take the form of suspensions,frozen formulations, centrifugation-concentrated frozen formulations,and the like.

In addition to the HGF gene introduced in this invention, it is possibleto use endogenous cardiac muscle protective factors or regenerationfactors against cardiac muscle. For example, it is reported thatfactors, such as TGF-β and heat shock protein (HSP) expressed highlyduring damage of the cardiac muscle, reduce myocardiopathy and areengaged in the repair of cardiac muscle. Therefore, it is possible touse the genes encoding them. Moreover, growth factors, such as EGF, arereported to repair cell damage in various tissues and genes encodingthem can be also used. In addition to these cardiac muscle protectivefactors and regeneration factors, factors related to protection andregeneration of the cardiac muscles can be utilized.

According to the invention, it is possible to deliver the protein ofinterest to damaged cells, such as cardiac muscle cells, by introducingan HGF gene, along or together with other genes, to the cardiac musclecell of the heart and highly expressing them. This enables activation ofrepair and regeneration of the damaged cardiac muscle and such, andrecuperation of the cardiac function involved in myocardiopathy. Hence,the gene therapy agent of this invention can be applied to patients withcritical cardiomyopathy, and offers remedy for patients for whom nooptions, other than heart transplantation, are left.

Moreover, the therapeutic agent of this invention can be applied notonly to patients with severe cardiomyopathy but also to patients withprogressive mild cardiomyopathy. It is applicable to patients with acardiac muscle disorder such as angina pectoris and heart failure aswell.

Proper methods and sites for administration adequate for the disease orsymptom to be treated are selected for the gene therapy agent of thisinvention. Cardiac muscle (affected part of the cardiac muscle) is apreferable administration site. As to the administration methods,parenteral administration methods are preferred.

Examples of parenteral administration methods include administration bynoninvasive catheter, noninvasive injector and so on. More preferred areadministration methods which utilize noninvasive catheter, noninvasiveinjector and such under the usage of echo. As a method using noninvasivecatheter, for example, methods like injecting HGF genes directly can beindicated.

Dosage of the therapeutic agent of this invention varies depending onthe symptoms of the patient but HGF genes 0.0001 mg to 100 mg,preferably about 0.001 to 10 mg per adult patients can be defined.

When the HVJ-liposome form is chosen, HGF genes of a range of about 1 toabout 4000 μg, preferably about 10 to about 400 μg per adult patient isselected.

The therapeutic agent of this invention is suited for administrationonce very few days or every few weeks, and administration once per weekis preferred.

Frequency of administration is to be selected depending on the symptomsof the patients. In compliance with the object of the treatment, pluraladministration is suitable, and preferably administration of 8 times canbe indicated.

Further to the present invention, a new gene therapy method andtherapeutic agent used therefor, including noninvasive administration oftherapeutically effective gene for the treatment of the disorder to theaffected tissue site under the usage of echo, is presented. That is, itwas revealed for the first time that effective treatments can beachieved visually by administering directly the gene to the affectedtissue under the usage of echo. According to the therapeutic treatmentof the invention, genes are administered noninvasively and thereforedesired genes can be administered as much as the condition demands,which is advantageous as compared to former methods. Gene therapymethods of this invention can be applied to any genes, in addition toHGF gene. This gene therapy method of the invention is particularlyeffective when applied to the affected site of cardiac muscle. Genesadministered in such situations include the HGF gene, TGF-β gene, HSPgene, VEGF gene, FGF gene, EGF gene and so on.

The present invention will now be specifically explained with referenceto the following examples. It should be noted, however, that the presentinvention is not limited by these examples in any way.

MATERIALS AND METHODS

Experimental Animals

Hamster model for cardiomyopathy (cardiomyopathy hamster; Bio14.6) waspurchased for Oriental Yeast.

HGF Gene

Human HGF gene was cloned from human HGF cDNA (Japanese Patent No.2777678) according to a conventional method and was inserted into theexpression vector pcDNA (Invitrogen).

Experimental Procedure

1. Reporter gene luciferase was introduced into the cardiomyopathyhamster by HVJ liposome under the usage of echo. A week later, theactivity of the luciferase was measured. Animals into which PBS wasintroduced alone under the usage of echo were used as the control.Luciferase activity was measured by a luminometer (LamatLB9507(BERTHOLO)).2. Under the usage of echocardiogram (MD500, YOKOKAWA-GE), HVJ-liposomeagent was injected into the abdominal lateral cardiac muscle of theheart of myocardiopathy hamster (12 weeks old) and was subjected tofollowing investigations:1) Density of blood capillary in the cardiac muscle was measured by ALP(alkaline phosphatase) staining and the result of the HGF gene wascompared to that of the control.2) Bloodstream of the heart to which HVJ-liposome was administered wasevaluated by laser Doppler imager (LDI) score and the result of the HGFgene was compared to that of the control.3) After Masson staining of the cardiac muscle, distribution density offibrosis was measured by computer analysis. Result of the HGF gene wascompared to that of the control.Reference 1Preparation of HVJ-Liposome Agent

10 mg Dried lipid (a 1:4.8:2 mixture of phosphatidyl serine,phosphatidyl choline and cholesterol) and 200 μl isotonic solution (137μM NaCl, 5.4 μM KCl, 10 μM Tris-HCl; pH7.6) containing HGF gene (100μg)-HMG1 (high mobility group 1 nuclear protein, 25 μg) was mixed and,by stirring vigorously with ultrasonication, liopsomes were formed.Purified Sendai virus (Z strain) was irradiated with UV (110erg/mm²/sec) for 3 minutes. Liposome suspension was mixed with Sendaivirus (HVJ), heated at 4° C. for 10 minutes, and then heated at 37° C.for 30 minutes. Free HVJ was discarded and thus obtained HVJ liposomeagent.

Reference 2

Measurement on Luciferase Activity

Liposome agent with 10 μg of luciferase gene was administered tohamsters (6 animals per group). A week later, luciferase activity wasmeasured. Results are shown in FIG. 1.

As shown in FIG. 1, high levels of luciferase activity were exhibited inthe heart. Thus, it was revealed that gene transfer under the usage ofecho is possible.

Experiment 1

Treatment of Myocardiopathy Hamster with HGF Gene

Liposome agent was injected into the abdominal lateral cardiac muscle ofthe heart of myocardiopathy hamsters (12 weeks old, 6 animals pergroup). A group of myocardiopathy hamsters (12 weeks old, 6 animals pergroup) to which liposome agent containing control vectors was injectedin the same manner was used as the control and untreated myocardiopathyhamsters (6 animals per group) were used as the untreated group. Thenliposome agents were injected once each week for 8 times. 8 weeks later,density of blood capillary in the cardiac muscle of the heart of the 20week old myocardiopathy hamsters was measured by ALP staining, andbloodflow was evaluated by the LDI score. After euthanization of thehamsters, the heart was extirpated and after Masson staining,distribution density of fibrosis was measured by computer analysis.

ALP staining revealed significant rise in blood capillary byangiogenesis in HGF gene treatment group. The results are shown in FIG.2.

Concerning LDI score, taking the control group as 100%, the HGF genetreatment group was 163±7%, which indicates significant increase inbloodflow. The results are shown in FIG. 3.

According to the analysis of Masson staining, significant decrease indistribution density of fibrosis was observed in HGF gene treatmentgroup. The results are shown in FIG. 4.

INDUSTRIAL APPLICABILITY

Therapeutic agents for myocardiopathy comprising an HGF gene of thisinvention induce angiogenesis of the affected part of cardiac muscle,increase bloodflow of the affected part while repressing and reducingfibrosis of the cardiac muscle it can repair the cardiac function.Moreover, therapeutic agents of this invention can be injectednoninvasively and accurately to the affected cardiac muscle layervisually under the usage of echo. Therefore, therapeutic agents of theinvention enable more effective treatment of myocardiopathy.

1. A method for treating a cardiac muscle disorder comprisingadministering a therapeutically effective amount of a nucleic acidmolecule encoding hepatocyte growth factor (HGF) directly to a part ofan affected abdominal lateral cardiac muscle of a mammal usingechocardiographic guidance without thoracotomy, wherein the nucleic acidmolecule is encapsulated in a Sendai virus (HVJ)-liposome and expressesan HGF protein that reduces fibrosis and/or promotes angiogenesis of thecardiac muscle, thereby treating the cardiac muscle disorder.
 2. Amethod for treating a cardiac muscle disorder comprising administering atherapeutically effective amount of a nucleic acid molecule encoding HGFdirectly into an abdominal lateral cardiac muscle of a mammal, whereinthe nucleic acid molecule is administered once a week for 8 weeks,wherein the nucleic acid molecule is encapsulated in a Sendai virus(HVJ)-liposome and expresses and HGF protein that reduces fibrosisand/or promotes angiogenesis of the cardiac muscle, thereby treating thecardiac muscle disorder.
 3. The method of claim 1, wherein the cardiacmuscle disorder is angina pectoris or heart failure.
 4. A method fortreating a cardiac muscle disorder comprising administering atherapeutically effective amount of a nucleic acid molecule encoding HGFdirectly into a abdominal lateral cardiac muscle of a mammal, whereinadministering comprises administering the nucleic acid molecule underechocardiographic guidance without thoracotomy through a catheter,wherein the nucleic acid molecule is encapsulated in a Sendai virus(HVJ)-liposome and expressions an HGF protein that reduces fibrosisand/or promotes angiogenesis of the cardiac muscle, thereby treating thecardiac muscle disorder.